Despite reductions in malaria cases across Latin America, malaria transmission hot spots remain heterogeneously distributed throughout Amazonia, virtually all of which are strongly linked to the primary Amazonian vector Anopheles darlingi. Malaria transmission in Amazonia is associated with rapid adaptation by An. darlingi to anthropogenically-driven environmental changes. This project will address three understudied facets of An. darlingi which will provide key details of how this vector maintains its dominant role in Amazonian malaria transmission dynamics by: 1) delineating the importance of anthropic breeding sites that lead to high mosquito productivity and dispersal near human habitation; 2) examining rapid adaptation in modified landscapes of both location (indoor/ outdoor) and nocturnal timing of biting behavior; and, 3) showing how this mosquito species? preferences/ changes interact with Plasmodium vivax asymptomatic persons to maintain hypoendemic transmission. First, we will evaluate the contribution of artificial vs. natural ponds to malaria transmission. In villages/towns with varying annual parasitic indices in malaria endemic regions of Peru and Brazil, we will census breeding sites near malaria-associated houses and characterize their physical parameters, and assess host availability in and around houses. We will identify where mosquitoes bite infected individuals by identifying patterns of mosquito movement, particularly by comparing larval genotypes in breeding site types with adults from houses. We expect a strong correlation of larval density in proximity with ?hot? (malarial) houses that experience the highest numbers of cases within village regardless of breeding site type. Second, based on our previous success in continuous laboratory propagation of An. darlingi in the Peruvian Amazon, we will colonize behaviorally distinctive (endophagic, exophagic) populations of An. darlingi and experimentally test each for vector competence using P. vivax and P. falciparum. We anticipate detecting two distinct populations, 1) wild, opportunistic mosquitoes in natural breeding sites, bloodseeking preferentially from ~6-9 PM outside houses; 2) anthropophilic mosquitoes that preferentially use artificial breeding sites, bloodseeking from ~3-6 AM inside houses. Third, we will quantify and compare the efficiency of P. vivax infection of An. darlingi under different, biologically-relevant conditions, using direct and standard membrane feeding assays. The following groups of subjects will be studied: symptomatic, microscopy positive; asymptomatic, microscopy positive; and asymptomatic, subpatent (microscopy negative, RT-PCR+), with asymptomatic, untreated subjects studied serially over short periods (diurnally over days) before treatment. These data will allow us to parameterize mathematical models of P. vivax transmission dynamics in quantifying biologically relevant factors such as fluctuations in parasitemia and gametocytemia over days and comparing circadian cycles. We expect to find that asymptomatic parasitemic individuals are quantitatively important reservoirs that maintain malaria transmission whether patent or subpatent.